Heterogeneous inseminate system

ABSTRACT

A first amount of sperm of a first animal and a second amount of sperm of a second animal of the same species, the first amount of sperm and the second amount of sperm sex-selected sperm of the same sex, useful in the in-vivo or in-vitro fertilization of an egg of a female animal of the same species of animal for the production of sex-selected embryos and sex-selected offspring.

This United States Patent Application is a continuation of U.S. patentapplication Ser. No. 15/332,123, filed Oct. 24, 2016, now U.S. Pat. No.10,492,896, issued Dec. 3, 2019, which is a continuation of U.S. patentapplication Ser. No. 13/702,986, filed Dec. 7, 2012, now U.S. Pat. No.9,474,591, issued Oct. 25, 2016, which is the United States NationalStage of International Patent Cooperation Treaty Patent Application No.PCT/US2011/001052, filed Jun. 8, 2011, which claims the benefit of U.S.Provisional Patent Application No. 61/353,140, filed Jun. 9, 2010, eachhereby incorporated by reference herein.

I. BACKGROUND

Artificial insemination of female animals and in-vitro fertilization ofoocytes with fresh or frozen-thawed semen have been applied to thereproduction of animals. According to the traditional theory andconventional procedures, a great number of sperm cells are needed toensure a successful fertilization.

However, semen from certain animals and sex-selected sperm which areproduced by separating X-chromosome bearing sperm cells fromY-chromosome bearing sperm cells into distinct subpopulations may beavailable only in limited supply. Additionally, the sperm of certainanimals and sex-selected sperm can have reduced fertility or cannotachieve the threshold of fertility in the context of conventionalprocedures for successful fertilization of eggs whether in-vitro orin-vivo.

Additionally, cryopreservation and thawing of semen and sex-selectedsperm can result in substantially reduced motility, percent intactacrosomes and post thaw survival time, all of which can militate againstsuccessful use of cryopreserved-thawed sperm for the in-vitro or in-vivofertilization of eggs of the same species of animal.

Additionally, certain animals, such as dairy cows, are known to havereduced fertility as compared to heifers. An adverse side effect ofacquiring certain genetic traits advantageous to milk production can beconcurrent with acquisition of genetic traits which lower fertility ofdairy cows. Moreover, as to the example of dairy cows, it may also bedesirable to increase the proportion of female offspring by the use ofsex-selected sperm. In order to successfully breed animals havingreduced fertility, especially in the instance of utilizing sex-selectedsperm to increase the number of female offspring, it would be useful tohave an inseminate, and a method of producing such inseminate, havingincreased fertility in order to increase pregnancy rates.

Therefore, a need exists for a method of increasing fertility of spermwithin an inseminate to overcome reduced fertility in inseminateswhether due to low numbers of sperm contained in the inseminate, lowfertility of sperm relating to animal to animal variation, the processof sex-selection, the low fertility of the female animal, or the like.

II. SUMMARY OF THE INVENTION

Accordingly, a broad object of the invention can be to provide aheterogeneous inseminate in which a first amount of sperm and a secondamount of sperm obtained from a corresponding first animal and a secondanimal are combined resulting in an increase in the fertility of atleast one of the first amount of sperm or the second amount of spermsuch that the eggs of an animal can be fertilized whether by in-vitro orin-vivo insemination with increased success rates as compared withconventional inseminates containing the sperm of only one animal.

Another broad object of the invention can be to provide a heterogeneousinseminate in which a first amount of sperm and a second amount of spermobtained from a corresponding first animal and a second animal arecombined, the second amount of sperm containing substantially fewersperm than the first amount of sperm, resulting in an increase infertility in the second amount of sperm such that the eggs of an animalcan be fertilized whether in-vitro or in-vivo by one of the sperm of thesecond amount of sperm with increased success rates as compared toconventional inseminates containing only a reduced number of sperm ofthe second animal, thereby increasing the number of embryos produced perunit of a rare genetic resource and can reduce the cost of embryoproduction.

Another broad object of the invention can be to provide a heterogeneousinseminate in which a first amount of sperm and a second amount of spermobtained from a corresponding first animal and a second animal arecombined, the first or the second amount of sperm or both beingsex-selected sperm, resulting in an increase in fertility of the firstor second amount of sex-selected sperm resulting in an increase infertility in the first or the second amount of sex-selected sperm orboth such that the eggs of an animal can be fertilized whether in-vitroor in-vivo by one of the sperm of the first or second amount ofsex-selected sperm with increased success rates as compared toconventional inseminates containing only sex-selected sperm of only oneanimal, thereby increasing the number of embryos produced per unit ofsex-selected sperm and can reduce the cost of embryo production.

Another broad object of the invention can provide a cryopreservedheterogeneous inseminate in which a first amount of sperm and a secondamount of sperm obtained from a corresponding first animal and a secondanimal are combined, the first or the second amount of sperm or bothbeing sex-selected sperm, resulting in an increase in fertility of thefirst or second amount of sex-selected sperm upon being thawed resultingin an increase in fertility in the first or the second amount ofsex-selected sperm or both such that the eggs of an animal can befertilized whether in-vitro or in-vivo by one of the sperm of the firstor second amount of sex-selected sperm with increased success rates ascompared to conventional cryopreserved inseminates containingsex-selected sperm of only one animal, thereby increasing the number ofembryos produced per unit of sex-selected sperm and can reduce the costof embryo production.

Naturally, further objects of the invention are disclosed throughoutother areas of the specification, drawings, photographs, and claims.

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram which shows a particular method of how to makeand use an embodiment of the inventive heterogeneous inseminate.

FIG. 2 is a block diagram which shows a particular method of how to makeand use an embodiment of the inventive heterogeneous inseminate.

FIG. 3 is an illustration which shows a particular method of producing asex-selected heterogeneous inseminate by using a flow cytometer in themethod shown in FIG. 1 or shown in FIG. 2 to separate X-chromosomehearing sperm cells from Y-chromosome bearing sperm cells.

FIG. 4 is a plot which shows the level of resolution achieved between anX-chromosome bearing population and a Y-chromosome bearing populationresulting from the separation of sperm in a first amount of spermobtained from a first male animal using a flow cytometer in accordancewith the method shown in FIG. 2.

FIG. 5 is a bivariate plot which shows separation of sperm of the firstamount of sperm obtained from a first animal separated into anX-chromosome bearing population and a Y-chromosome bearing populationusing flow cytometer in accordance with the method shown in FIG. 2.

FIG. 6 is a plot which shows the level of resolution achieved between anX-chromosome bearing population and a Y-chromosome bearing populationresulting from the separation of sperm of a first amount of spineobtained from a first male animal combined with a second amount of spermobtained from a second male animal using flow cytometry in accordancewith the method shown in FIG. 1.

FIG. 7 is a bivariate plot which shows the level of resolution achievedbetween an X-chromosome bearing population and a Y-chromosome bearingpopulation resulting from the separation of sperm of a first amount ofsperm obtained from a first animal combined with a second amount ofsperm obtained from a second animal using flow cytometry in accordancewith the method shown in FIG. 1.

FIG. 8 is an illustration which shows an enlarged portion the bivariateplot shown in FIG. 7.

IV. MODE(S) FOR CARRYING OUT THE INVENTION

Generally, a heterogeneous inseminate produced by combining a firstamount of sperm obtained from a first animal with a second amount ofsperm of a second animal of the same species useful in the in-vitro orin-vivo fertilization of an egg of a female animal of the species forthe production embryos and offspring. Specifically, a first amount ofsperm of a first animal and a second amount of sperm of a second animalof the same species, the first amount of sperm and the second amount ofsperm sex-selected sperm of the same sex, useful in the in-vivo orin-vitro fertilization of an egg of a female animal of the same speciesof animal for the production of sex-selected embryos and sex-selectedoffspring.

For the purposes of this invention, the term “animal” means a species ofmammal, ayes, or fish from which semen containing an amount of sperm isproduced by a male animal of the species and an egg is produced by thefemale animal of the species and a mammal, without limiting the generalforgoing statement, can be a species of: horse, cattle, camel, deer,pig, sheep, goat, dog, cat, lion, whale, dolphin, porpoise, seal, hare,rabbit, elephant, mouse, rat, or the like, and a fish, without limitingthe general forgoing statement, can be a species of: salmon, tuna,sturgeon, halibut, catfish, or the like, and a bird, without limitingthe general forgoing statement can be a species of: chicken, turkey,eagle, falcon, ostrich, emu, duck, goose, or the like.

For the purposes of this invention the term “semen” means seminal fluidwhich may contain sperm (also referred to as “spermatozoa”) secreted bythe gonads of a male animal which can be collected from the male animalby a variety of methods such as use of an artificial vagina, manualmanipulation of the penis, electrical manipulation of the anus, or thelike.

For the purposes of this invention the term “sperm” means the haploidcell that is the gamete of a male animal which may join an egg (alsoreferred to as “ovum”) to produce a zygote and broadly encompassesinfertile sperm, sperm having a comparably lesser or a comparablygreater fertility between a first amount of sperm obtained from a firstanimal and a second amount of sperm obtained from a second animal andwhich may be obtained in the form of a raw ejaculated semen, frozensemen, as sperm separated from the semen and contained in an extender ordiluent, or as sex-selected sperm.

For the purposes of this invention the term “sex-selected sperm” meanssperm which have been separated, regardless as to the method ofseparation, into subpopulations containing X-chromosome bearing spermand Y-chromosome bearing sperm having a purity in the range of about 70percent (“%”) and about 100%.

For the purposes of this invention, the term “inseminate” is intended tobroadly encompass an amount of sperm whether contained in semen or otherextender or diluent which can be utilized to fertilize the eggs of afemale animal whether in-vitro or in-vivo and without limitation to thegeneral forgoing statement can be an amount of sperm whether or notsex-selected introduced or injected into the reproductive tract of afemale animal whether by artificial insemination procedures orotherwise, or utilized in in-vitro fertilization procedures.

For the purposes of this invention, the term “heterogeneous inseminate”means an inseminate in the form of a combination of a first amount spermobtained from a first male animal and a second amount of sperm obtainedfrom a second male animal, the first animal and the second male animalbeing of the same species; however, the invention is not so limited anda plurality of amounts of sperm obtained from a corresponding pluralityof male animals of the same species can be combined to produce variousembodiments of the heterogeneous inseminate.

For the purposes of this invention, the term “sex-selected heterogeneousinseminate” means a heterogeneous inseminate having the first amount ofsperm obtained from a first male animal and the second amount of spermobtained from a second male animal (or a plurality of amounts of spermobtained from a plurality of animals) which are processed, whether priorto combination or after combination, such that the first amount of spermand the second amount of sperm (or the plurality of amounts of sperm)contained in a heterogeneous inseminate are sex-selected sperm.

For the purposes of this invention, the term “fertility” means thefertilization rate determined for an artificial insemination procedureas the number of pregnancies achieved within a population of inseminatedfemale animals divided by the total number inseminated female animalsand multiplied by 100 or determined for in-vitro fertilization procedureas the number of embryos (greater than or equal to two cells) divided bythe sum of the embryos and oocytes recovered and multiplied by 100.

For the purposes of this invention, the term “motility” means movingsperm in an inseminate or sample of sperm.

For the purposes of this invention, the term “infertile” means as to anyparticular procedure incapable of fertilizing an egg of a female animal.

Now referring to primarily to FIG. 1, a plurality of amounts of spermcells (1) can be obtained from a corresponding plurality of male animals(2), each of the plurality of male animals (2) of the same species. Theterm “plurality of male animals” (2) can encompass two, three, four,five, six, or a greater number of male animals (2). The plurality ofamounts of sperm cells (1) can be combined to achieve a heterogeneousinseminate (3). The heterogeneous inseminate (3) can be divided toproduce a number of artificial insemination dosages (4) which can as toparticular embodiments be contained one each in a corresponding numberartificial insemination straws (5). One or more of the artificialinsemination doses (4) can be used for artificial insemination (6) of afemale animal (7) of the same species for the production of in-vivoembryos (8) whether as single embryo pregnancies to generate offspring(9) for meat or animal replacement or multiple embryo pregnancies formultiple ovulation embryo transfer (“MOET”) by subsequent flushing of aplurality of in-vivo embryos (8) from the female animal (7). As to otherembodiments, the heterogeneous inseminate (3) can be divided or used asa number of in-vitro fertilization doses (11) for in-vitro fertilization(12) of one or more eggs (13) obtained from a female animal (7) toproduce a plurality of in-vivo (8).

Again referring primarily to FIG. 1, while a plurality of amounts ofsperm (1) from a corresponding plurality of male animals (2) is shown; aparticular illustrative example useful in understanding the numerous andvaried embodiments of the invention can include a first amount sperm(14) obtained from a first male animal (15) and a second amount of sperm(16) obtained from a second male animal (17). The first male animal (15)and the second male animal (17) being of the same species. The firstamount of sperm (14) obtained from the first male animal (15) can becombined with the second amount of sperm (16) obtained from the secondmale animal (17) to produce a particular embodiment of the heterogeneousinseminate (3).

As to particular embodiments of the heterogeneous inseminate (3), thefirst amount of sperm (14) can be an amount of infertile sperm (18)while the second amount of sperm (16) can have an initial level offertility (19) outside of the heterogeneous inseminate (3). Uponcombination of the first amount of sperm (14) (infertile sperm (18))with the second amount of sperm (16), as concentration of the firstamount of sperm (14) increases in the heterogeneous inseminate (3) ascompared to the concentration of the second amount of sperm (16), thesecond amount of sperm (16) can exhibit by comparison to the initiallevel of fertility (19) an increased level of fertility (20) within theheterogeneous inseminate (3). Accordingly, the first amount sperm (14)can be sufficient in number or concentration within the heterogeneousinseminate (3) such that, the second amount of sperm (16) achieves bycomparison to the initial level of fertility (19) an increased level offertility (20).

As to other particular embodiments of the heterogeneous inseminate (3),the first amount of sperm (14) outside the heterogeneous inseminate (3)can have greater fertility than the initial level of fertility (19) ofthe second amount of sperm (16) outside of the heterogeneous inseminate(3). In combination, the second amount of sperm (16) can exhibit anincreased level of fertility (19) within the heterogeneous inseminate(3). Accordingly, the heterogeneous inseminate (3) can afford asubstantial advantage over conventional inseminates when the secondamount of sperm (16) can confer desirable genetic traits but has aninitial level of fertility (19) outside of the heterogeneous inseminate(3) which may be unsuitable for use as an artificial insemination dosage(4) for the artificial insemination (6) of a female animal (7) or may beunsuitable for use as an in-vitro fertilization dose (11) for in-vitrofertilization (12) of eggs (13) obtained from a female animal (7). Thecomparably lesser initial level of fertility (19) of the second amountof sperm (16) outside of the heterogeneous inseminate (3) as compared toincreased level of fertility (20) inside of the heterogeneous inseminate(3) may be due to a number of factors. For example, there may be alesser concentration of sperm outside of the heterogeneous inseminate(3), or the first amount of sperm (14) having greater initial level offertility (19) may contribute, supplement or afford within theheterogeneous inseminate (3) a physical stimulus or chemical stimulusthat acts upon the second amount of sperm (16) to generate the increasedlevel of fertility (20) within the heterogeneous inseminate (3), or thesecond amount of sperm (16) may exhibit an increased level of motility(60) within the heterogeneous inseminate (3) as compared to outside ofthe heterogeneous inseminate (3), such greater motility can act toincrease fertility of the second amount of sperm (16) in theheterogeneous inseminate (3).

Now referring primarily to FIG. 1 and FIG. 2, embodiments of theheterogeneous inseminate (3) can further include a first amount of sperm(14) and a second amount of sperm (16) (or a plurality of amounts ofsperm (1)) from a corresponding first male animal (15) and a second maleanimal (17) (or plurality of male animals (2)) of the same species inwhich the first amount of sperm (14) or the second amount of sperm (16)or both the first amount of sperm (14) and the second amount of sperm(16) (or plurality of amounts of sperm (1)) can be sex-selected sperm(21).

Now referring primarily to FIG. 1, as to particular embodiments, thesex-selection process (22) for production of sex-selected sperm (21) canoccur after combining the first amount of sperm (14) with the secondamount of sperm (16) (or after combining a plurality of amounts of sperm(1)) to achieve the heterogeneous inseminate (3). After combination, theheterogeneous inseminate (3) can then undergo the sex selection process(22) resulting in a sex-selected heterogeneous inseminate (23)containing X-chromosome bearing sperm (24) and a sex-selectedheterogeneous inseminate (25) containing Y-chromosome bearing sperm(25). The sex-selected heterogeneous inseminate (23) can containX-chromosome bearing sperm (24) of the first amount of sperm (14) andthe second amount of sperm (16) (or the plurality of amounts of sperm(1)) in similar proportion or concentration as in the originalheterogeneous inseminate (3) to afford the substantial advantagesabove-described.

Similarly, the sex-selected heterogeneous inseminate (23) can containY-chromosome bearing sperm (25) of the first amount of sperm (14) andthe second amount of sperm (16) (or plurality of amounts of sperm (1))in similar proportion or concentration to the original heterogeneousinseminate (3) to afford the substantial advantages above-described. Thesex-selected heterogeneous inseminate (23) containing X-chromosomebearing sperm (24) or the sex-selected heterogeneous inseminate (23)containing (Y-chromosome bearing sperm (25) can be used in-vitro orin-vivo to fertilize the eggs (13) of a female animal (7) of the samespecies to produce sex-selected in-vivo embryos (26) and sex-selectedoffspring (9) or produce sex-selected in-vitro embryos (27) of apre-selected sex. While FIGS. 1 and 2, show a sex-selected heterogeneousinseminate (23) containing only X-chromosome bearing sperm (24) of twoor more male animals (2) being used for in-vivo (6) and in-vivo (12)insemination of eggs (13); the invention is not so limited, and as abovedescribed embodiments of the sex-selected homogeneous inseminate (23)can include the X-chromosome bearing sperm (24) of two or more maleanimals (2) or the Y-chromosome bearing sperm (25) of two or more maleanimals (2).

Now referring primarily to FIG. 2, as to particular embodiments, thesex-selection process (22) for production of sex-selected sperm (21) canoccur prior to combining the first amount of sperm (14) with the secondamount of sperm (16) (or prior to combining the plurality of amounts ofsperm (1)) to produce a sex-selected heterogeneous inseminate (23). Asto these embodiments, each of the first amount of sperm (14) and thesecond amount of sperm (16) (or the plurality of amounts of sperm (1))separately undergo the sex selection process (22). The first amount ofsperm (14) can be separated into a subpopulation of X-chromosome bearingsperm (24) and into a subpopulation of Y-chromosome bearing sperm (25).Similarly, the second amount of sperm (16) (or each of the plurality ofamounts of sperm (1)) can be separated into isolated subpopulationsrespectively containing X-chromosome bearing sperm (24) and Y-chromosomebearing sperm (25). An amount of the X-chromosome bearing sperm (24)obtained from the first amount of sperm (14) can be combined with anamount of the X-chromosome bearing sperm (24) obtained from the secondamount of sperm (16) (or the X-chromosome bearing sperm (24) of aplurality of amounts of sperm (1) can be combined) to provide thesex-selected heterogeneous inseminate (23) containing X-chromosomebearing sperm (24). Similarly, an amount of Y-chromosome bearing sperm(25) obtained from the first amount of sperm (14) can be combined withan amount of the Y-chromosome bearing sperm (25) obtained from thesecond amount of sperm (16) (or the Y-chromosome bearing sperm of aplurality of amounts of sperm (1) can be combined) to provide asex-selected heterogeneous inseminate (23) containing Y-chromosomebearing sperm (25).

The amount of the X-chromosome bearing sperm (24) obtained from thefirst amount of sperm (14) can be adjusted in relation to the amount ofthe X-chromosome bearing sperm (24) obtained from the second amount ofsperm (16) (or the X-chromosome bearing sperm obtained from each of theplurality of amounts of sperm (1) can be adjusted in relation to eachother) to achieve a desired concentration or proportion of X-chromosomebearing sperm (24) from the first amount of sperm (14) and the secondamount of sperm (16) (or the plurality of amounts of sperm (1)) in thesex-selected heterogeneous inseminate (23) to afford the substantialadvantages above-described.

Similarly, the amount of the Y-chromosome bearing sperm (25) obtainedfrom the first amount of sperm (14) can be adjusted in relation to theamount of the Y-chromosome bearing sperm (25) obtained from the secondamount of sperm (16) (or the Y-chromosome bearing sperm obtained fromeach of the plurality of amounts of sperm (1) can be adjusted inrelation to each other) to achieve a desired concentration or proportionof Y-chromosome bearing sperm (25) from the first amount of sperm (14)and the second amount of sperm (16) (or the plurality of amounts ofsperm (1)) in the sex-selected heterogeneous inseminate (23) to affordthe substantial advantages above-described.

The sex-selected heterogeneous inseminate (23) containing X-chromosomebearing sperm (24) or the sex-selected heterogeneous inseminate (23)containing Y-chromosome bearing sperm (25) (or a particular proportionof X-chromosome bearing sperm (24) to Y-chromosome bearing sperm (25))can be used for in-vitro fertilization (12) or in-vivo artificialinsemination (6) to fertilize the eggs (13) of a female animal of thesame species to produce in-vivo sex-selected embryos (26) or in-vitrosex-selected embryos (27) of a pre-selected sex.

In regard to the increased level of fertility (20), particularembodiments of the invention can include a first amount of sperm (14)which outside of the heterogeneous inseminate (3) can have an initiallevel of fertility (19) of zero or nearly zero for a particularapplication, which can be due to insufficient numbers of sperm,insufficient motility of sperm, or insufficient physical or chemicalstimuli which can be afforded within the heterospermic inseminate (3).Accordingly, once the threshold as to numbers of sperm, motility ofsperm or the physical or chemical stimuli is achieved within theheterogeneous inseminate (3), the increased level of fertility (20) ofthe second amount of sperm (16) can be sufficient to allow successfulfertilization of an egg (13) of a female animal (7) for that particularapplication, which would otherwise be unsuccessful or have a lesserfertility rate.

Understandably, the initial level of fertility (19) can be any level offertility within a particular application, and the increased level offertility (20) can be a comparable increase of fertility for thatparticular application. One measure by which initial level of fertility(19) and increased level of fertility (20) can be assessed is by measureof the initial level of motility (60) of the second amount of sperm (16)(or of any of the plurality of amounts of sperm (1)) outside of theheterogeneous inseminate (3) and the increased level of motility (61) ofthe second amount of sperm (16) (or of any of the plurality of amountsof sperm (1)) within the heterogeneous inseminate (3). As to embodimentsof the inventive heterogeneous inseminate (3) or sex-selectedheterogeneous inseminate (23) the increase in the motility of the secondamount of sperm (16) within the heterogeneous inseminate (3) (orsex-selected heterogeneous inseminate (23)) as compared to outside ofthe heterogeneous inseminate (3) can be in the range of between about 1%and about 30%. With respect to sex-selected heterogeneous inseminates(23) as one example, the initial level of motility (60) of sex-selectedsperm (21) outside of the sex-selected heterogeneous inseminate (23)shortly after separation by a sex-selection process (22), such as flowcytometry, can be at or below 45% as compared to the increased level ofmotility (61) of a substantially identical sample of sex-selected sperm(21) within the heterogeneous inseminate (3) of about 60% to about 70%.

Now referring primarily to FIG. 3, without limiting the forgoing, aparticular process for separating X-chromosome bearing sperm (24) fromY-chromosome bearing sperm (25) is shown. A flow cytometer (28) can beconfigured to differentiate and the first amount of sperm (14) and thesecond amount of sperm (16) (where prior combined or separately) basedon the amount of deoxyribonucleic acid (“DNA”) (29) within the spermhead (30). Typically, a sheath fluid source (31) delivers a fluid stream(32) in which a plurality of sperm cells (33) delivered from a spermcell source (34) can be entrained. The fluid stream (32) entraining aplurality of sperm cells (33) can be oscillated by a nozzle (35) togenerate a plurality of droplets (36) below the nozzle (35). Each of theplurality of droplets (36) can entrain one of the plurality of spermcells (33). An illumination source (37), such as a laser (38), can emita beam of light (39), or a plurality of beams of light can be generatedby utilizing a plurality of illumination sources (37) or by utilizing abeam splitting element (40) (the beam splitting element shown but notthe plurality of beams of light) which can be focused incident upon eachof the plurality of sperm cells (33) entrained in the fluid stream (32)below the nozzle (35) through an optical element (41), either as asingle beam of light (39) or a plurality of beams of light, whether atthe same or different wave lengths. Characteristics of the beam of light(39) can be altered by incidence upon each one of the plurality of spermcells (33) within the fluid stream (32), or an emission (42) can begenerated by incidence of the beam of light (39) upon ligands (43), suchas fluorescent materials, or the like, bound to the DNA (29) of each oneof the plurality of sperm cells (33). The beam(s) of light (39) havingaltered characteristics or the emission (42) can be received by a singleor a plurality of detectors (44) which can generate a signal (45) foranalysis by a computer implemented program (46) to differentiate eachone of the plurality of sperm cells (33) correspondingly entrained ineach one of the plurality of droplets (36) based upon one or a pluralityof sperm cell characteristics (47). Each differentiated one of theplurality of sperm cells (33) can be separated based upon the presenceor absence of one or a plurality of the analyzed sperm cellcharacteristics (47) and collected in a corresponding one of a pluralityof collection elements (48). The flow cytometer (28) can further includea droplet charge generator (49) which induces a positive or negativecharge in each one of the plurality of droplets (33) and a dropletdeflector (50) which acts upon each one of the charged plurality ofdroplets (36) to establish a trajectory to the proper one of theplurality of collection elements (48). Each of the plurality ofcollection elements (48) thereby contains a subpopulation of theplurality of sperm cells (33) which can be predominantly X-chromosomebearing sperm (24) or Y-chromosome bearing sperm (25). The purity level(%=100 times the result of X or Y divided by the total of X and Y) ofthe resulting subpopulation of X-chromosome bearing sperm (24) or thesubpopulation of Y-chromosome bearing sperm (25) can exceed 70%, 80%,90% or can be of even greater purity depending upon the operatingparameters of the flow cytometer (28) are adjusted; however, the puritylevel can be adjusted to be a greater or a lesser percent purityconsistent with any particular application. Non-limiting examples ofconventional methods of using a flow cytometer (28) to sort theplurality of sperm cells (33) suitable for use in the production of athe heterogeneous inseminate (3) are described in U.S. Pat. Nos.5,135,759; 6,372,422; 7,195,920 and 7,169,548, each hereby incorporatedby reference in the entirety herein; however, the invention is not solimited and any method of producing an enriched subpopulation ofX-chromosome bearing sperm (24) or an enriched subpopulation ofY-chromosome bearing sperm (25) can be utilized.

Now referring primarily to FIGS. 2, 4 and 5, a univariate plot (orhistogram) (51) (FIG. 4) and a bivariate plot (52) (FIG. 5) can begenerated during use of a flow cytometer (28) to separate or sort afirst amount of sperm (14) or a second amount of sperm (16) or each of aplurality of amounts of sperm (1) from a corresponding first male animal(15), second male animal (17) or plurality of male animals (2) intosubpopulations of X-chromosome bearing sperm (24) and Y-chromosomebearing sperm (25) prior to being combined to achieve an embodiment ofthe sex-selected heterogeneous inseminate (23) as above described (seealso FIG. 2). The histogram (51) (FIG. 4) and the bivariate plot (52)(FIG. 5) show that the mixture of X-chromosome bearing sperm (24) andY-chromosome bearing sperm (25) in each of a first amount of sperm (14)or second amount of sperm (16) or plurality of amounts of sperm (1) canbe resolved into separate corresponding subpopulations of X-chromosomebearing sperm (24) and subpopulations of Y-chromosome bearing sperm(25).

Now referring primarily to FIGS. 6 through 8, a univariate plot orhistogram) (51) (FIG. 6) and bivariate plot (52) (FIG. 7) (see also FIG.8 showing an enlarged portion of bivariate plot shown in FIG. 7) can begenerated during the use of a flow cytometer (28) to separate or sortX-chromosome bearing sperm (24) from Y-chromosome bearing sperm (25) ina heterogeneous inseminate (3) prior achieved by combination of a firstamount of sperm (14) with a second amount of sperm (16) or bycombination of a plurality of amounts of sperm (1) from a correspondingfirst animal (15), second animal (17) or plurality of animals (2) of thesame species as above described (also refer to FIG. 1). Because of thedifferences in the same sperm characteristics (47) between a firstamount of sperm (14) obtained from a first animal (15) and a secondamount of sperm (16) obtained from a second animal (17), or between aplurality of amounts of sperm (1) obtained from a correspondingplurality of animals (2) of the same species, the separation, selection,or sorting (depending upon the method) of the X-chromosome bearing sperm(24) from the Y-chromosome bearing sperm (25) in a heterogeneousinseminate (3) may not have been achieved prior to the instantinvention.

For the purposes of this invention, the term “extender” refers to asolution that comes in contact with the plurality of sperm cells (1) orthe heterogeneous inseminate (3), or sex-selected sperm (21) orsex-selected heterogeneous inseminate (23), typically for the purpose ofdilution or as a cryoprotectant. Typical examples of an extender (53)can contain one or more of: sodium citrate,Tris[hydroxymethyl]aminomethane (also referred to as “TRIS”), TES(N-Tris [Hydroxymethyl]methyl-2-aminoethanesulfonic acid), monosodiumglutamate, HEPES medium such as HEPES buffered medium, HEPES bufferedbovine gamete medium and particularly HBGM3 and can further containcryoprotectants such as glycerol, dimethyl sulfoxide, ethylene glycol,propylene glycol; other organic substances such as egg yolk, an egg yolkextract, milk, a milk extract, casein, albumin, lecithin, bovine serumalbumin, cholesterol; sugars such as the monosacharides, glucose,fructose, or mannose; detergents such as sodium dodecyl sulfate;antioxidants such butylated hydroxytoluene; capacitation facilitatorssuch as alpha amylase, beta amylase, or beta glucuronidase; antibioticssuch as tylosin, gentamicin, lincomycin, spectinomycin, linco-spectin (acombination of lincomycin and spectinomycin), penicillin, streptomycin,and ticarcillin; flow cytometer sheath fluids; and specifically withoutlimiting the forgoing, particular extenders (53) referred to below asTRIS-A (54) and TRIS-B (54); although the inventive heterogeneousinseminate (3) or sex-selected heterogeneous inseminate (23) or methodsof using the heterogeneous inseminate (3) or sex-selected heterogeneousinseminate (23) are not limited by the working examples which use TRIS-A(54) or TRIS-B (55) as an extender (53) to dilute concentration of theplurality of sperm cells (1), the sex-selected sperm cells (21) orembodiments of the heterogeneous inseminate (3) or sex-selectedheterogeneous inseminate (23) which are diluted by use of TRIS-A (54) orthe use TRIS-B (55) as a cryoprotectant (56). The use of TRIS-A (54) orthe use of TRIS-B (55) is described in detail as an illustrative exampleof the numerous and varied extenders that can be used with embodimentsof the invention as diluents and cryoprotectants.

For the purposes of this invention the term “TRIS-A” refers to anextender (53) having the formulation in Table 1.

TABLE 1 TRIS-A Extender. TRIS 200 mM Citric Acid  65 mM Fructose  56 mMEgg Yolk* 20% by volume Hydrochloric Acid addition to pH 6.8 *Egg Yolkcan be clarified by winterization process to remove certain particulatesand fats.

The formulation of TRIS-A set out in Table 1 can from application toapplication of the invention be modified to increase viability or reducedamage to the plurality of sperm cells (1), the heterogeneousinseminates (3) or sex-selected heterogeneous inseminates (23) and theabove formulation is provided as a non-limiting example of a numerousand wide variety of similar extenders (53) which are suitable for use inmaking or using the embodiments of the invention. See also, Yassen, A.M. and Foote, R. H., Freezability of Bovine Spermatozoa in Tris-BufferedYolk Extenders Containing Different Levels of Tris, Sodium, Potassiumand Calcium Ions, J. Dairy Science, Vol. 50, No. 6, 887-892 (1966),hereby incorporated by reference in the entirety herein. Extenders (53)in general and specifically the particular extender TRIS-A (54) canfurther include one or more antibiotics (57) as above described orconsistent with animal health regulations of any particularjurisdiction. Also, the description of TRIS-A (54) is not intended to belimiting with respect to the wide variety of extenders (53) which can beutilized in making and using certain embodiments of the invention, asdescribed above.

For the purposes of this invention the term “TRIS-” refers to anextender having the formulation in Table 2.

TABLE 2 TRIS 200 mM Citric Acid  65 mM Fructose  56 mM Egg Yolk* 20% byvolume Glycerol 12% by volume Hydrochloric Acid addition to pH 6.8 *EggYolk can be clarified by winterization process to remove certainparticulates and fats.

The formulation of TRIS-B set out in Table 2 can from application toapplication of the invention be modified to increase viability or reducedamage to the plurality of sperm cells (1), the heterogeneousinseminates (3) or sex-selected heterogeneous inseminates (23) and theabove formulation is provided as a non-limiting example of a numerousand wide variety of similar extenders (53) which are suitable for use inmaking or using the embodiments of the invention. Again see for example,Yassen, A. M. and Foote, R. H., Freezability of Bovine Spermatozoa inTris-Buffered Yolk Extenders Containing Different Levels of Tris,Sodium, Potassium and Calcium Ions, J. Dairy Science, Vol. 50, No. 6,887-892 (1966). Extenders (53) in general and specifically theparticular extender TRIS-B (55) can further include one or moreantibiotics (57) as above described or consistent with animal healthregulations of any particular jurisdiction. Additionally, while TRIS-B(55) of the particular formulation set out in Table 2 uses glycerol as acryoprotectant (56) the invention is not so limited. Also, thedescription of TRIS-B is not intended to limiting with respect to thewide variety of extenders (53) which can be utilized in making and usingcertain embodiments of the invention, as described above.

Again referring primarily to FIGS. 1 and 2, as to particularembodiments, the plurality of sperm cells (1), the first amount of sperm(14) and the second amount of sperm (16) (whether or not sex-selectedsperm (21)), the resulting heterogeneous inseminate (3) or sex-selectedheterogeneous inseminate (23) can further include an amount of seminalfluid (58), which may be may be cell free semen (59) prepared by removalof entrained sperm (1) which may be diluted with an extender (53).Removal of entrained sperm (1) from the seminal fluid (58) can beachieved by centrifugation of semen at about 500×g for about 15 minutes.The seminal fluid (58) decanted and centrifuged a second time at 500×gfor about 15 minutes.

Again referring to FIGS. 1 and 2, embodiments of the homogeneousinseminate (3) or sex-selected homogeneous inseminate (23) can furtherinclude a particular dosage form. As one non-limiting example, thedosage form for artificial insemination (6) of cattle can be aone-quarter cubic centimeter (0.25 cc) artificial insemination straw (5)which contains the heterogeneous inseminate (3) or sex-selectedheterogeneous inseminate (23). While the Figures only show asex-selected heterogeneous inseminate (23) contained in the artificialinsemination straw (5); the invention is not so limited and dosage formswhether in the form of an artificial insemination straw (5) or otherwisecan contain a heterogeneous inseminate (3), a sex-selected heterogeneousinseminate (23), or can contain a combination of both a heterogeneousinseminate (3) and sex-selected heterogeneous inseminate (23).

Again referring to FIGS. 1 and 2, dosage forms of the heterogeneousinseminate (3) and the sex-selected heterogeneous inseminate (23) can beproduced by similar steps or procedures as conventional insemination orsex-selected insemination dosage forms; however, the use of theheterogeneous inseminate (3) or sex-selected heterogeneous inseminate(23) can by comparison provide artificial insemination dosages (4) orin-vitro fertilization dosages (11) which exhibit an increased level ofmotility (61) or have an increased level of fertility (20).

Now referring primarily to FIG. 1, as one non-limiting example, a bovineembodiment of a sex-selected heterogeneous inseminate (23) can beproduced by obtaining a first amount of sperm (14) (whether fresh orcryopreserved) of a first male animal (15) and obtaining a second amountof sperm (16) (whether fresh or cryopreserved) of a second male animal(17) of the same species of bovine male animal (or by obtaining aplurality of amounts of sperm (1) from a plurality of male animals (2)of the same species). The first amount of sperm (14), the second amountof sperm (16), or the plurality of amounts of sperm (1) can each beseparately suspended in an amount of TRIS-A (54) (or other extender(53)) and then centrifuged in range of about 500 rounds per minute(“rpm”) and about 5,000 rpm for a period of time in a range of betweenabout one minute and about ten minutes. The supernatant can be decantedfrom each of the amounts of sperm (14)(16)(1). Each of the amounts ofsperm (14)(16)(1) can be extended with TRIS-A (54) (or other extender(53)) to achieve a concentration which allows each the extended firstamount of sperm (14), second amount of sperm (16), or plurality ofamounts of sperm (1) to be combined to produce a heterogeneousinseminate (3) each of the first amount of sperm (14), the second amountof sperm (16), or each of a plurality of amounts of sperm (1) at adesired concentration within the heterogeneous inseminate (3).

As a non-limiting example, the concentration of the first amount ofsperm (14) and the second amount of sperm (16) within the heterogeneousinseminate (3) can be about 75 million sperm per milliliter. Theconcentration of the first amount of sperm (14) in the heterogeneousinseminate (3) can be about 55 million sperm per milliliter and theconcentration of the second amount of sperm (16) in the heterogeneousinseminate (3) can be about 20 million sperm per milliliter; however theinvention is not so limited and the concentration of the first amount ofsperm (14) or the second amount of sperm (16) or a plurality of amountsof sperm (1) within the heterogeneous inseminate (3) can be at anypredetermined concentration depending upon the application, which as tocertain embodiments can provide about equal concentration of each of aplurality of amounts of sperm (1) from a corresponding plurality ofanimals (2) or can provide unequal concentration as to a plurality ofamounts of sperm (1) from a corresponding plurality of animals (2).

The heterogeneous inseminate (3) can then be incubated in about 38micromolar Hoechst 33342 at about 75 million sperm/ml in the extender(53) for 1 hour at about 34° C.; although other stains or stainingconditions can be utilized, to achieve staining of the DNA of the firstamount of sperm (14), the second amount of sperm (16), or the pluralityof amounts of sperm (1) within the heterogeneous inseminate (3) forseparation into subpopulations containing X-chromosome bearing sperm(24) and Y-chromosome bearing sperm (25), as above described orotherwise. As an example, the stained sperm contained in theheterogeneous inseminate (3) can be interrogated by laser excitation at351 and 364 nm at 150 mW and based on the difference in fluorescentemission be separated into subpopulations of X-chromosome bearing sperm(24) and Y-chromosome bearing sperm (25) using a MOFLO flow cytometer(28) (or other makes or models of flow cytometers or multiple flowchannel apparatus). The subpopulations of X chromosome-bearing sperm(24) and Y-chromosome bearing sperm in of about 90% purity as verifiedby resort analysis can be collected at between about 1,500 livesperm/second and about 5,000 live sperm in/second into 2-ml Eppendorftubes containing 100 micromolar Cornell Universal Extender (CUE) with20% egg yolk.

Collected sex-selected sperm (21) can be centrifuged at about 600×g for10 min and resuspended in TRIS B (55) to a concentration of about 2million motile sex-selected sperm/ml to provide an embodiment of asex-selected heterogeneous inseminate (23). The sex-selectedheterogeneous inseminate (23) can be cooled to about 5° C. over about 75minutes and artificial insemination dosages (4) transferred into 0.25-ccartificial insemination straws (5) (about 150 microliters to about 200microliters per straw). Straws can be cryopreserved, as above describedby conventional procedures, or transported at about 3° C. to about 5° C.for use in artificial insemination (6) of female animals (7). Artificialinsemination dosages (4) and control dosages can be inseminated usingside-opening blue sheaths (IMV), one half of each straw into eachuterine horn of female animal (7) of the same bovine species (total ofabout 3.5×10⁵ sex-selected sperm per artificial insemination dosage orabout 250,000 sex-selected sperm from the first amount of sperm (14) andabout 50,000 sex-selected sperm from the second amount of sperm (16)).Understandably, the respective concentration of sex-selected sperm (21)in an artificial insemination dosage (4) between a first amount of sperm(14), a second amount of sperm (16), or a plurality of amounts of sperm(1) can be adjusted by adjusting the initial concentration of thesex-selected insemination (23) prior to division into the correspondingplurality of artificial insemination dosages (4) other dosage forms.

Now referring primarily to FIG. 2, as a general non-limiting example, abovine embodiment of a sex-selected heterogeneous inseminate (23) can beproduced by obtaining a first amount of sperm (14) as sex-selected spermcells (21) (whether fresh or cryopreserved) of a first male animal (15)of a bovine species. The first amount of sperm (14) can be suspended inan amount of TRIS-A (54) (or other extender (53)) and then centrifugedin range of about 500 rounds per minute (“rpm”) and about 5,000 rpm fora period in a range of between about one minute and about ten minutes.The supernatant can be decanted. The first amount of sperm (14) can beextended with TRIS-A (54) extender (or other extender (53)) to achieve aconcentration of the extended first amount of sperm (14) of about fourtimes greater than the expected concentration of the second amount ofsperm (16), or other concentration depending upon the application. Thefour times concentration of the first amount of sperm (14) in TRIS-A(54) (or other extender (53)) can be for example a concentration in arange of about 3.2 million sperm cells per milliliter and about 128million sperm cells per milliliter.

The extended first amount of sperm (14) (sex-selected sperm (21)) can becooled to a temperature in a range of about 4 degrees Celsius (“° C.”)and about 5° C. The cooled extended first amount of sperm (14) can beheld at this temperature to allow the membranes of the first amount ofsperm (14) to move toward equilibrium or equilibrate with the TRIS-A(typically a period of about 90 minutes or as to certain embodiments notless than 90 minutes). The cooled extended first amount of sperm (14)can be held in this condition not to exceed a length of time in whichthe sperm cells (14) remain viable or capable fertilizing an egg (13) ofa female animal (7) of the same species as the first and second maleanimals (15)(17). Typically, the period of time held will not exceed 12hours.

A second amount of sperm (16) as sex-selected sperm cells (21) (fresh orcryopreserved) can be obtained of a second male animal (17) of the samebovine species. The second amount of sperm (16) can be suspended in anamount of TRIS-A (54) (or other extender (53)) and then centrifuged inrange of about 500 rounds per minute (“rpm”) and about 5,000 rpm for aperiod in a range of between about one minute and about ten minutes. Thesupernatant can be decanted and the pellet containing the second amountof sperm (16) can be suspended in an amount of TRIS-A (54) to achieve aconcentration of the second amount of sperm (16) of about one-fourththat of the first amount of sperm (14). Accordingly, the second amountof spine (16) as a non-limiting example can have a concentration in arange of about 800,000 sperm per milliliter and about 32 million spermper milliliter depending upon various factors such as the species of thesecond male animal (17), the scarcity of the sperm, the fertility of thesperm, the dosage form, the method of insemination (whether artificialinsemination (6) or in-vitro fertilization (12)), whether for multipleovulation embryo transfer or single ovulation single embryo production,the female animal (7) of the same species as the first and second maleanimal (15)(17) being inseminated, or the like.

The extended second amount of sperm (16) can be cooled to a temperaturein a range of about 4° C. and about 5° C. The cooled extended secondamount of sperm (16) can be held at this temperature to allow themembranes of the second amount of sperm (16) to move toward equilibriumor equilibrate with the TRIS-A (54) (typically a period of about 90minutes or as to certain embodiments not less than 90 minutes).

About equal volumes of the cooled extended first amount of sperm (14)(sex-selected sperm (21)) and the cooled extended second amount of sperm(16) (sex-selected sperm (21)) can be combined to achieve two timesgreater concentration of the first amount of sperm (14) with respect tothe final concentration of the second amount of sperm (16) contained inthe resulting heterogeneous sex-selected inseminate (23).

To the resulting sex-selected heterogeneous inseminate (23) about anequal volume of TRIS-B (55) (containing between about 12% and 20%glycerol) can be added and the combination cooled to temperature in arange of about 4° C. and about 5° C. The cooled extended sex selectedheterogeneous inseminate (23) can be held at this temperature to allowthe membranes of the sperm cells (1)(21) to move toward equilibrium orequilibrate with the TRIS-B (55) (typically a period in the range ofabout 30 minutes and about 90 minutes or as to certain embodiments notless than 90 minutes). The cooled sex selected heterogeneous inseminate(23) can be held in this condition not to exceed a length of time inwhich the sex-selected sperm cells (21) remain viable or capablefertilizing an egg (13) of a female of the same species as the maleanimals (2).

The cooled sex-selected heterogeneous inseminate (23) can be handled bynormal procedures utilized for producing artificial insemination doses(6). Accordingly, as to certain embodiments, the cooled sex selectedheterogeneous inseminate (23) can be aliquoted into 0.25 cc artificialinsemination straws (5) (about 150 microliters to about 200 microlitersper straw). The 0.25 cc artificial insemination straw (5) can contain anamount of the sex-selected heterogeneous inseminate (23) which containssex-selected sperm (21) from the second amount of sperm (16) in therange of about 50,000 and about 2 million and from the first amount ofsperm (14) in the range of about 200,000 and about 8 million. However, agreater or lesser number of the first amount of sperm (14) and thesecond amount of sperm (16) can be included a 0.25 cc artificialinsemination straw (5) by adjusting the initial concentrations of thefirst amount of sperm (14) and the second amount of sperm (16) with thetotal number of sperm in each artificial insemination dosage (4) of theinstant example being in the range of about 250,000 and about 10 millionsex-selected sperm (21).

The sex-selected heterogeneous inseminate (23) can be used forartificial insemination (6) to fertilize the egg(s) (13) of a femaleanimal (7) of the same species as the first and second male (15)(17) forthe production of sex-selected in-vivo embryo (26) whether as singleembryo pregnancies to generate offspring (9) for meat or animalreplacement or multiple embryo pregnancies for MOET by subsequentflushing of a plurality of sex-selected embryos (10). Alternately, thesex-selected heterogeneous inseminate (23) can be used in-vitrofertilization procedures (12) to fertilize eggs (13) of obtained from afemale animal (7) of the same species to produce in vitro sex-selectedin-vitro fertilized embryos (27).

The use of heterogeneous sex-selected inseminate (23) can result inincreased fertility rates. As one non-limiting example, where aplurality of amounts of sperm (1) are processed in accordance witheither the method shown in FIG. 1 or FIG. 2 and as described above toproduce a sex-selected heterogeneous inseminate (23), the sex-selectedheterogeneous inseminate (23) can have an increased level of fertility(20) overall as compared to any initial level of fertility (19) for anyone of the plurality of amounts of sperm (1) outside of the sex-selectedheterogeneous inseminate (23).

As a second non-limiting example, where a plurality of amounts of sperm(1) from a corresponding plurality animals (2) of the same species areprocessed as above described to provide a sex-selected heterogeneousinseminate (23) which includes at least one amount of sex-selected sperm(21) having a comparably greater initial level of fertility (19) to oneor more amounts of sex-selected sperm (23) having a lesser initial levelof fertility (19) outside of the sex-selected heterogeneous inseminate(23), the one or more of the amounts of sex-selected sperm (21) withinthe sex-selected heterogeneous inseminate (23) will exhibit an increasedlevel of fertility (20) which can exceed the initial level of fertility(19) of the amount of sex-selected sperm having the greater initiallevel of fertility (19).

As a third non-limiting example, the seminal fluid (58) of one or moreamounts of sex-selected sperm (21) which exhibit a comparably greaterinitial level of fertility (19) outside of the sex-selectedheterogeneous inseminate (23) can be stored as an amount of cell freesemen (59) and added back into the sex-selected heterogeneous inseminate(23) processed in accordance with either the method shown in FIG. 1 orFIG. 2 and as described above to produce a sex-selected heterogeneousinseminate (23). The amount of cell free semen (59) in a sex-selectedheterogeneous inseminate (23) can be in the range of zero cell freesemen to about 2 parts cell free semen (59) to one part sex-selectedheterogeneous insemination (23). The concentration of the sex-selectedsperm (21) can be adjusted as above-described to afford the desiredconcentration of sex-selected sperm (21) in an artificial inseminationdosage (4) or an in-vivo insemination dosage (11).

For the purpose of the present invention, the term “combination orcombining or combined” refers to any method of putting two or morematerials together. Such methods include, but are not limited to,mixing, commingling, incorporating, intermingling, stirring,integrating, or the like.

For the purposes of the present invention, ranges may be expressedherein as from “about” one particular value to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “a spermcell” refers to one or more of sperm cells. As such, the terms “a” or“an”, “one or more” and “at least one” can be used interchangeablyherein unless otherwise indicated. According to the present invention,an isolated material or particle is removed from its natural milieu;however, an isolated material does not necessarily reflect the extent towhich the material or particle has been purified.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. Theinvention involves numerous and varied embodiments of a reduced doseinseminate (3) and methods of making and using such a reduced doseinseminate (3). As such, the particular embodiments or elements of theinvention disclosed by the description or shown in the figuresaccompanying this application are not intended to be limiting, butrather exemplary of the numerous and varied embodiments genericallyencompassed by the invention or equivalents encompassed with respect toany particular element thereof. In addition, the specific description ofa single embodiment or element of the invention may not explicitlydescribe all embodiments or elements possible; many alternatives areimplicitly disclosed by the description and figures.

It should be understood that each element of an apparatus or each stepof a method may be described by an apparatus term or method term. Suchterms can be substituted where desired to make explicit the implicitlybroad coverage to which this invention is entitled. As but one example,it should be understood that all steps of a method may be disclosed asan action, a means for taking that action, or as an element which causesthat action. Similarly, each element of an apparatus may be disclosed asthe physical element or the action which that physical elementfacilitates. As but one example, the disclosure of a “flow sorter”should be understood to encompass disclosure of the act of “flowsorting” whether explicitly discussed or not and, conversely, were thereeffectively disclosure of the act of “flow sorting”, such a disclosureshould be understood to encompass disclosure of a “flow sorter” and evena “a means for flow sorting.” Such alternative terms for each element orstep are to be understood to be explicitly included in the description.

In addition, as to each term used it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood to beincluded in the description for each term as contained in the RandomHouse Webster's Unabridged Dictionary, second edition, each definitionhereby incorporated by reference.

Thus, the applicant(s) should be understood to claim at least: i) eachof the heterogeneous inseminate herein disclosed and described, ii) therelated methods disclosed and described, iii) similar, equivalent, andeven implicit variations of each of these devices and methods, iv) thosealternative embodiments which accomplish each of the functions shown,disclosed, or described, v) those alternative designs and methods whichaccomplish each of the functions shown as are implicit to accomplishthat which is disclosed and described, vi) each feature, component, andstep shown as separate and independent inventions, vii) the applicationsenhanced by the various systems or components disclosed, viii) theresulting products produced by such systems or components, ix) methodsand apparatuses substantially as described hereinbefore and withreference to any of the accompanying examples, x) the variouscombinations and permutations of each of the previous elementsdisclosed.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed or deemed to be admitted as prior art withrespect to the invention.

The claims set forth in this specification, if any, are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice-versa as necessary to define the matter for whichprotection is sought by this application or by any subsequentcontinuation, division, or continuation-in-part application thereof orto obtain any benefit of reduction in fees pursuant to, or to complywith the patent laws, rules, or regulations of any country or treaty,and such content incorporated by reference shall survive during theentire pendency of this application including any subsequentcontinuation, division, or continuation-in-part application thereof orany reissue or extension thereon.

In addition, the claims set forth below are intended to describe themetes and bounds of a limited number of the preferred embodiments of theinvention and are not to be construed as the broadest embodiment of theinvention or a complete listing of embodiments of the invention that maybe claimed. The applicant does not waive any right to develop furtherclaims based upon the description set forth above as a part of anycontinuation, division, or continuation-in-part, or similar application.

We claim:
 1. A composition, comprising: a first amount of sperm of ananimal, said first amount of sperm containing X-chromosome bearing spermcells and Y-chromosome bearing sperm cells; and a second amount of spermof said animal, said second amount of sperm comprising an isolatedsubpopulation of X-chromosome bearing sperm cells or an isolatedsubpopulation of Y-chromosome bearing sperm cells of said animal, saidfirst amount of sperm of said animal combined with said second amount ofsperm of said animal, wherein concentration of said first amount ofsperm in said composition is sufficient to increase motility orfertility of said second amount of sperm in said composition.
 2. Thecomposition of claim 1, wherein said first amount of sperm has a greaterconcentration in said composition than said second amount of sperm insaid composition.
 3. The composition of claim 1, wherein said firstamount of sperm comprises infertile sperm.
 4. The composition of claim1, wherein said first amount of sperm or said second amount of spermcomprises frozen-thawed sperm.
 5. The composition of claim 1, furthercomprising admixing an amount of cell free seminal fluid with said firstamount of sperm and said second amount of sperm.
 6. The composition ofclaim 1, wherein said animal is selected from the group consisting of: abovine, an aves, and a fish.
 7. The composition of claim 1, wherein saidanimal comprises a bovine mammal and further comprising establishing aconcentration of said second amount of sperm of said animal in saidcomposition at about 2×10⁴ sperm per milliliter to about 4×10⁶ sperm permilliliter.
 8. The composition of claim 7, wherein said concentration ofsaid first amount of sperm of said animal in said composition is atabout 2×10⁷ sperm per milliliter to about 8×10⁷ sperm per milliliter. 9.The composition of claim 8, further comprising a dosage form of saidcomposition having a volume of about one-quarter milliliter to about onemilliliter.
 10. The composition of claim 9, further comprising anartificial insemination straw containing said dosage form of saidcomposition.
 11. The composition of claim 10, wherein said dosage formof said composition is frozen in said artificial insemination straw. 12.The composition of claim 1, wherein said isolated subpopulation ofX-chromosome bearing sperm cells or said isolated subpopulation ofY-chromosome bearing sperm cells of said second amount of sperm has anon-naturally occurring ratio of X-chromosome bearing sperm orY-chromosome bearing sperm.
 13. The composition of claim 12, whereinsaid first amount of sperm comprises an isolated subpopulation ofX-chromosome bearing sperm cells or an isolated subpopulation ofY-chromosome bearing sperm cells.
 14. The composition of claim 13,wherein said isolated subpopulation of X-chromosome bearing sperm cellsor said isolated subpopulation of Y-chromosome bearing sperm cells ofsaid first amount of sperm and said second amount of sperm each have anon-naturally occurring ratio of X-chromosome bearing sperm orY-chromosome bearing sperm.
 15. The composition of claim 14, whereinsaid isolated subpopulation of X-chromosome bearing sperm cells or saidisolated subpopulation of Y-chromosome bearing sperm cells of said firstamount of sperm or said second amount of sperm contain DNA binding anamount of stain.
 16. The composition of claim 1, wherein said firstamount of sperm comprises only infertile sperm.